Phosphoinositide 3-kinases (PI 3-kinases) are critical regulators of proliferation, motility, and apoptosis. The Class IA PI 3-kinase is a dimer containing distinct regulatory (p85) and catalytic (p110) subunits, both of which are frequently mutated in human cancer. While a large amount of energy in academia and industry has been applied to the study of these enzymes, there are critical aspects of PI 3-kinase regulation that remain poorly understood. For example, although the p110 catalytic subunit has been crystallized, there is little information as to how the distinct domains of p85 are oriented in space, or how mutants of p85 lead to a loss of PI 3-kinase regulation. A specific role of p110 catalytic subunit in the proliferation and tumorigenesis of PTEN null prostate cancer cells has been identified, but the mechanisms that regulate p110 in these cells are not clear. Although novel mutations of p85 have been identified in colon cancer and glioblastoma, the effects of these mutations on tumorigenesis and metastasis in vivo are not known. The current proposal uses both biochemical and genetic approaches to address fundamental questions concerning PI 3-kinase signaling in cancer. Aim 1 uses single molecule FRET measurements to produce a spatial map of the p85 regulatory subunit, and to determine how the binding of PI 3-kinase activators alters p85 conformation and interactions with the p110 catalytic subunit. Aim 2 examines the mechanism of p110 activation by G subunits. Sites of p110-G interactions will be defined using genetic and biochemical methods. These sites will then be mutated, in order to test the requirement for G binding in signaling by p110 in PTEN null prostate cancer cells. Aim 3 will study the signaling by p85 mutants in colon cancer and glioblastoma cell lines, the tumor types in which the recently described mutants were identified. Lentivirus methods will be used to knock down endogenous p85 and rescue with wild type or mutant p85. The effects of these mutants on in vitro cell growth and motility, and in vivo tumorigenesis and metastasis, will be studied. This proposal will define novel aspects of PI 3-kinase signaling and regulation, and will provide a better understanding of structural features that could be exploited as drug targets.